1., Specificity and Epitope Mapping of Four Monoclonal Antibodies. against SARS-CoV Nucleocapsid Protein

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1 VIROLOGICA SINICA May 2006 SARS-CoV N * 1** ** , , Specificity and Epitope Mapping of Four Monoclonal Antibodies against SARS-CoV Nucleocapsid Protein WANG Jian-wei 1* WANG Yan-bin 1 CHANG Zhao-rui 1 YAN Ke-xia 1 TAN Wen-jie 1 QU Jian-guo 1 XIA Ning-shao 2* RUAN Li 1 HUNG Tao 1 (1. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing , China 2. The Research Center for Molecular Virology of Fujian Province, The Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen , China) Abstract: In order to characterize the specificity of the monoclonal antibodies (McAbs) against SARSassociated coronavirus (SARS-CoV) nucleocapsid protein and to identify the epitopes recognized by the McAbs the nucleocapsid proteins of human coronavirus OC43 (HCoV-OC43) and 229E (HCoV-229E) was expressed in E.coli. The specificities of four McAbs (1-1C2, 2-2E5, 1-1D6, and 2-8F11) were examined by Western blotting as well as indirect fluorescence assay. Twelve different recombinant truncated N proteins were then used to identify the epitopes recognized by the McAbs by Western blotting. The results showed that: (1) McAbs 1-1C2, 2-2E5, and 1-1D6 recognized neither human coronviruses HCoV-OC43 and HCoV-229E nor their nucleocapsid proteins, implying a possible specificity of these 3 McAbs to SARS-CoV; (2)The epitopes recognized by McAbs 2-8F11, 2-2E5, and 1-1D6 were located between the 30th and 60th amino acid (a.a.) residues of the SARS-CoV N protein, while the epitopes recognized by the McAb1-1C2 were located between 170th and 184th a.a. residues. The identification of the specific epitopes of SARS-CoV N protein is paramount for the immunological characterization, the development of accurate immunological assay as well as for exploring the pathogenesis of SARS-CoV. Key words SARS-associated coronavirus (SARS-CoV); Nucleocapsid protein; Monoclonal antibody; Epitope SARS-CoV N E.coli 229E HCoV-229E OC43 HCoV-OC4 N Western blotting 4 SARS-CoV N 1-1C2 1-1D6 2-8F11 2-2E5 HCoV-OC43 HCoV-229E N 12 SARS-CoV N N 1-1C2 1-1D6 2-2E5 HCoV-OC43 HCoV-229E N SARS-CoV N 2 2-8F11 1-1D6 2-2E5 * AA EPISARS ** Corresponding authors. wangjw28@ vip.sina.com, nsxia@jianxian.xmu.edu.cn

2 SARS-CoV N aa C2 SARS-CoV N aa SARS-CoVN SARS (SARS-CoV) R511 A (2006) SARS SARS (SARS-associated coronavirus SARS-CoV) 1 RT-PCR RT-PCR RNA [1~3] 2 N ELISA [4] 3 ELISA N [5] SARS IgM 7d 14d [6] RT-PCR RNA, SARS RT-PCR SARS-CoV N OC43 229E [7,8] SARS-CoV SARS-CoV SARS-CoV N (monoclonal antibody, McAb) McAb SARS-CoV SARS-CoV IgG Pierce FITC IgG Sigma 6 His Sigma HCoV-229E HCoV-OC4 HCoV-229E HCoV-OC43 SARS-CoV N McAb 1-1C2 1-1D6 2-8F11 2-2E5 [9] E.coli DH5α BL21 DE3 pet-30a Novagen SARS-CoV N E.coli [10] SARS-CoV N PN160 aa1-160 PN194 aa1-194 PN267 aa1-267 PN301 aa1-301 PN363 aa1-363 PN170 aa1-170 PN184 aa1-184 PN185 aa PN155 aa PN125 aa PN95 aa PN75 aa [11] E OC43 N GenBank 229E AF OC43 AY E OC43 N 5 3 Sal I Xho I 229E OC43 N pet30a Sal I Xhol I E.coli BL21(DE3) LB 37 1: ml LB 37 2h 1mmol/L IPTG 5h 1.3 [12] SDS-PAGE 5% 4 IgG 1.4 HCoV-229E HCoV-OC43 RDa (m.o.i.=5) [13] 30% - SARS-CoV N McAb HcoV-229E HCoV-OC4 FITC IgG HCoV-229E HCoV-OC43 N GenBank HCoV-229E HCoV-OC43 229E OC43 N pet-30a E.coli BL21 DE3 IPTG SDS-PAGE 54kD(HCoV-OC43 N) 48KD(HCoV-229E N) 1A 2

3 ,. 209 Western blotting 6 His Tag 6 His 1B 1 HCoV-229E HCoV-OC43 N E.coli Fig. 1 Analysis of the recombinant nucleocapsid proteins of HCoV-OC43 and HCoV-229E expressed in E. coli. A: SDS-PAGE. B: Western blotting. 1: OC43 N protein; 2: 229E N protein; C: Negative control; M: Protein markers 2.2 SARS-CoV N HCoV- 229E HCoV-OC43 SARS-CoV N McAb HCoV-229E HCoV-OC43 HCoV-229E HCoV-OC43 4 SARS-CoV N McAb 1-1D6 2-2E5 2-8F11 1-1C2 HCoV-229E HCoV- OC43 2 HCoV-229E HCoV-OC43 A B SARS-CoV N HCoV-229E HCoV-OC43 RD-A Fig. 2 Indirect immunofluorescence assay of HCoV-229E and HCoV-OC43 in RDa cells with different monoclonal antibodies against SARS-CoV nucleocapsid protein A: HCoV-229E. B: HCoV-OC43. 1, Positive control; 2, 1-1C2; 3, 1-1D6; 4, Negative control; 5, 2-2E5; 6, 2-8F11.

4 SARS-CoV N HCoV- 229E HCoV-OC43 N SARS-CoV N McAb HCoV-229E HCoV-OC43 N E.coli SARS-CoV HCoV-229E HCoV-OC43 N SDS-PAGE 4 McAb Western blotting HCoV-229E HCoV-OC43 N 1-1D6 2-2E5 1-1C2 HCoV-229E N 48 KD HCoV-OC43 N 54 KD 2-8F11 HCoV-229E N HCoV-OC43 N 4 McAb SARS-CoV N 46 KD SARS-CoV N SARS-CoV N 4 Western blotting McAb PN160 aa PN194 aa PN267 aa PN301 aa PN363 aa D6 2-2E5 2-8F C2 PN194 PN267 PN301 PN363 4 PN D6 2-2E5 2-8F11 aa C2 aa McAb 8 SARS-CoV N 4 Western blotting PN160 aa1-160 PN170 aa1-170 PN184 aa C2 PN185 aa PN155 aa PN125 aa PN95 aa PN75 aa D6 2-2E5 2-8F11 1-1C2 PN184 aa D6 2-2E5 2-8F11 PN185 aa D6 2-2E5 2-8F11 aa C2 aa SARS-CoV N HCoV-229E HCoV-OC43 N Western blotting Fig.3 Reaction of HCoV-229E and HCoV-OC43 nucleoca Psid protein with anti-sars-cov nucleocapsid protein monoclonal antibodies by Western blotting. 1: SARS-CoV nucleocapsid protein Positive control ; 2: HCoV-OC43 nucleocapsid protein; 3: HCoV-229E nucleocapsid protein; C: Negative control. 2.4 SARS-CoV N 1 4 SARS-CoV N Table 1 Reaction of different nucleocapsid protein fragments with 4 monoclonal antibodies by Western blotting Truncated proteins Monoclonal antibodies Name Domain (aa) 1-1D6 2-2E5 2-8F11 1-1C2 PN160 1~ PN170 1~170 / / / - PN184 1~184 / / / + PN194 1~ PN267 1~ PN301 1~ PN363 1~ PN185 30~ / PN155 60~ / PN125 90~ / PN95 120~ / PN75 140~ / Notes: +: Positive; -: Negative; /: Not Done. 3 N SARS-CoV 22.6%~33.0% [14]

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